Counting single RNA molecule probes

This software is intended to assist with the enumeration of single molecule RNA probes. It operates on microscope data files, eventually outputting an annotated image showing a projection of the data with suggested cell boundaries and probe counts for those cells.

Installation notes for Mac

Ensure that you have Xcode installed, for example by running gcc in the terminal. Alternatively, you can use the app store.

gcc

Install Homebrew.

ruby -e "$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/master/install)"

Install freetype and libtiff using Homebrew.

brew install freetype
brew install libtiff

Install virtualenv using easy_install (virtualenv allows you to create a virtual Python environment).

sudo easy_install virtualenv
virtualenv env

Source the virtual environment (note the . at the start of the line).

. ./env/bin/activate

Install Python dependencies into the virtual environment.

pip install numpy
pip install pillow
pip install scipy
pip install six
pip install "scikit-image==0.10.1"
pip install freetype-py
pip install libtiff

Install BioFormats bftools

If you are working with czi file you may need BioFormats less than version 4.4.4, e.g. 4.4.2

The Python image analysis code works on Tiff files. We therefore need to convert the microscopy data using the BioFormats bfconvert tool.

The BioFormats tools require Java, which needs to be installed. Download the installer from the Oracle website. https://www.java.com/en/download/mac_download.jsp?locale-=en

Install the Java JDK. Download the installer form the Oracle website. http://www.oracle.com/technetwork/java/javase/downloads/jdk8-downloads-2133151.html

Install both of them.

Download the bftools.zip file from:

http://www.openmicroscopy.org/site/support/bio-formats5.0/users/comlinetools/

Place the downloaded bftools directory where you want it. For example:

mkdir ~/tools
mv ~/Downloads/bftools ~/tools/

Add the bftools directory to your $PATH environment variable.

export PATH=$PATH:~/tools/bftools

Image analysis

Download FISHCount software

Download the FISHcount software zip file from GitHub.

https://github.com/JIC-CSB/FISHcount/archive/master.zip

Go into the FISHCount directory (e.g. ~/Downlaods/FISHcount).

cd ~/Downloads/FISHcount

Convert microscopy images to tiff files

Make sure that the bftools directory is in your path.

export PATH=$PATH:~/tools/bftools

Run the unpack.py script.

python scripts/protoimg/unpack.py lif_input_dir tiff_output_dir

Where lif_input_dir is the directory with the original lif files and tiff_output_dir is the directory to where the tiff files will be written.

Do the image analysis

Make sure that the Python virtual environment has been sourced.

. ./env/bin/activate

Run the analysis on the image directory of interest and specify an output directory to store any generated images.

python ./scripts/count_probes.py tiff_output_dir/image_of_interest_dir output_dir

Note that the file of most interest is output_dir/annotated_projection.png.